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Muscle lymphocytic infiltrates in thymoma-associated myasthenia gravis are phenotypically different from those in polymyositis
Neuromuscular Disorders 17 (2007) 935–942 www.elsevier.com/locate/nmd
Muscle lymphocytic infiltrates in thymoma-associated myasthenia gravis are phenotypically different from those in polymyositis Josef Zamecnik a
d
a,*
, Dan Vesely a, Branislav Jakubicka a, Libuse Simkova b, Jiri Pitha c, Jan Schutzner d, Radim Mazanec e, Hannes Vogel f
Department of Pathology and Molecular Medicine, Charles University, 2nd Medical Faculty and Faculty Hospital Motol, V Uvalu 84, 150 06 Prague, Czech Republic b Department of Neurology, Charles University, 1st Medical Faculty, Prague, Czech Republic c Department of Neurology, Charles University, 3rd Medical Faculty, Prague, Czech Republic 3rd Department of Surgery, Charles University, 1st Medical Faculty and Faculty Hospital Motol, Prague, Czech Republic e Department of Neurology, Charles University, 2nd Medical Faculty, Prague, Czech Republic f Department of Pathology, Stanford University School of Medicine, Palo Alto, CA, USA Received 10 April 2007; received in revised form 21 May 2007; accepted 25 May 2007
Abstract The aim of the study is to provide evidence that the lymphocytic infiltration of myasthenia gravis (MG) muscle do not represent a true autoimmune myositis, rather an infiltration by naive lymphocytes derived from lymphocyte-rich thymomas. Muscle biopsies from 179 patients with pure MG, 6 thymoma patients without MG and 15 patients with definite polymyositis were analyzed. In 18 patients with MG (all associated with lymphocyte-rich thymomas) and in two thymoma patients without MG, lymphocytic infiltrates were identified in muscles. By use of immunohistochemistry, we demonstrated that the lymphocytes in MG differ from those in polymyositis, being mature but in contrast to polymyositis naive CD45RA+ T lymphocytes. We suggest that the lymphocytic infiltrates in patients with MG and thymoma represent an infiltration of muscle by thymoma-derived mature but naive T cells. The finding of CD8+CD45RA+ lymphocytes in muscle may signify an underlying thymoma and should not be misdiagnosed as polymyositis. 2007 Elsevier B.V. All rights reserved. Keywords: Myasthenia gravis; Polymyositis; Thymoma
1. Introduction The principal disease mechanism in myasthenia gravis (MG) is a humoral immune attack upon the acetylcholine receptor (AChR) leading to the characteristic defect of neuromuscular transmission [1,2]. Focal collections of mononuclear cells in MG muscles, so-called ‘‘lymphorrhages’’, were reported over 100 years ago [3]; however, their significance and pathogenic role has been a challenging issue in
*
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neuropathology for decades. The proposed role in causing MG by acting at the neuromuscular junction (NMJ) [4,5] could not be subsequently confirmed [6] and the presence of lymphocytes has been often interpreted as a coincidental inflammatory myopathy – polymyositis (PM). On the other hand, an increasing body of evidence suggests that the ‘‘PM’’ occurring in MG patients is clinically not of the same type as a classical PM [7], aside from the fact that PM is traditionally regarded as a disturbance of cellular immunity in striated muscle [8]. Our interest in this problem began with the case of a patient with the ambiguous presentation of muscle weakness with non-conclusive initial EMG findings, borderline
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serum levels of anti-AChR autoantibodies and normal serum levels of creatine kinase (CK), referred for a muscle biopsy. Although the infiltration of muscle by CD8+ lymphocytes was interpreted as a PM, the patient soon developed classic MG; a subsequent CT scan of mediastinum revealed a thymic tumor and after thymectomy the patient remained treated with pyridostigmin without immunosuppression and his status improved. This case prompted our systematic study of lymphocytic infiltrates in muscle tissue from MG patients. We present results indicating that true autoimmune PM may rarely develop in MG patients, but that it should be distinguished from the more frequent occurrence, i.e. the infiltration by naive lymphocytes derived from lymphocyte-rich thymomas. 2. Materials and methods 2.1. Patients 179 thymectomized MG patients (78 males and 101 females, median age 49 years) with available follow-up data for at least 1 year after thymectomy were retrospectively included in the study. The diagnosis of MG was based on a typical clinical pattern, a positive edrophonium test and neurophysiological investigations with decrement at repetitive stimulation and/or increased jitter at single-fiber EMG [2], in addition to the presence of an increased serum level of anti-AChR antibodies in the vast majority of cases (91.1%, n = 163). All patients underwent CT scan of the anterior mediastinum, which showed a suspicious thymic tumor in 33 cases (18.4%). In each case, the presence of the thymoma was verified by subsequent surgery and the diagnosis was confirmed by the histopathological examination. The clinical data were collected both at the time of initial diagnosis, shortly preoperatively, then one year after thymectomy and onwards each year to the end of followup (from 12 up to 165 months; median ± standard deviation, 27 ± 4.9 months). We recorded clinical symptoms, serum levels of CK and transaminases, erythrocyte sedimentation rate, C-reactive protein (CRP) level and blood cell counts. A standard transsternal thymectomy was performed in all the patients (T-3a according to the Myasthenia Gravis Foundation of America (MGFA) thymectomy classification [9]). All thymuses were examined histologically. Thymomas were typed according to the recent WHO classification of thymomas [10] into following categories: type A (spindle cell or medullary thymoma), type B (lymphocytes-rich, cortical thymoma) and mixed type AB thymoma. No other thymic tumors were observed in our series. The immunophenotype of the lymphocytes in thymomas was analyzed using the same antibodies as in the muscle (see below). Except for MG patients, muscle biopsies of 6 thymoma patients (median age 41 years) without any signs of muscle involvement at the time of surgery with available follow-up data entered into the study. Muscle biopsies of 15 patients (median age 57 years) with definite isolated PM based on the clinical findings,
EMG and confirmed by muscle biopsy with significant response to immunosuppressive therapy were examined as a control group. 2.2. Muscle biopsy – histology, histochemistry and immunohistology An excision from the sternothyroid muscle (approx. 10 · 5 · 5 mm in size) was obtained from all the thymectomized patients from the margin of the operational field at the onset of thymectomy (informed consent of the patient was obtained in each case). In patients with PM lower limb muscles were biopsied. Muscle tissues were snap frozen in isopentane (2-methylbutane, Sigma–Aldrich Co.) cooled in liquid nitrogen. Cryosections were examined by routine hematoxylin–eosin (H&E) staining and a conventional spectrum of histochemical reactions (described elsewhere [11]), from which the visualization of neuromuscular junctions using reactions for non-specific esterase was relevant for the study. In each case, we searched for lymphocytes in the serial cryosections by use of both the routine H&E stain and the immunohistochemical reactions with anti-CD45 (leukocyte common antigen, LCA) antibody. Provided that lymphocytes were found, the contiguous sections were subjected to immunohistochemistry for establishing their phenotype using antibodies against the antigens CD20, CD3, CD8, CD4 and TdT (terminal deoxynucleotidyltransferase). The CD45RA antigen (present on B lymphocytes and on mature but naive T lymphocytes) was also investigated. The number of macrophages was assessed using the anti-CD68 immunohistochemistry. Helpful tools in diagnosis of inflammatory myopathies, i.e. expression of HLA-I antigens and of membrane attack complex of complement (MAC, C5b-9) [12], were also examined. In addition to the non-specific esterase, immunohistochemistry for acetylcholinesterase was performed for localization of NMJs. Further details concerning the antibodies used in the study are indicated in Table 1. Following blocking of endogenous peroxidase activity, 4 lm thick cryosections were incubated overnight at 4C with appropriate primary antibodies. The antigen–antibody complexes were visualized by biotin–streptavidin detection systems (LSAB2 System or ChemMate Detection kit, Dako Co.). Chromogenic development was performed using 3,3 0 -diaminobenzidine (DAB, Fluka Chemie GmbH.). All sections were counterstained slightly with Harris’ hematoxylin. Positive and negative controls were provided with each assay. 2.3. Evaluation of MG severity and the outcome of MG patients Since the recently recommended Quantitative MG score for Disease Severity (QMGSDS) [9] could not be obtained from all patients at the onset of the disease in this retrospective study, the initial severity of MG was graded using a modified Osserman’s scale [13,14]:
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Table 1 Antibodies, dilutions, pretreatment and detection kits used for the immunohistochemical studies Antigen
Antibody; clone
Source
Dilution, pretreatment, detection kit
CD45 (LCA) CD20 CD3 CD4 CD8 CD45RA CD68 AChE HLA-I C5b-9 (MAC) TdT
mm; 2B11 + PD7/26 mm; L26 rp; NCL-CD3p mm; 4B12 mm; C8/144B mm; 4KB5 mm; PG-M1 mm; HR2 mm; W6/32 mm; aE11 mm, SEN28
Dako, Co. Dako, Co. Novocastra, Novocastra, Dako, Co. Dako, Co. Dako, Co. Novocastra, Dako, Co. Dako, Co. Novocastra,
1:1000, MWa 1:300, MWa 1:200, MWb 1:20, MWb 1:200, MWb 1:75, MWa 1:100, MWa 1:40a 1:900a 1:100a 1:100, MWa
Ltd. Ltd.
Ltd.
Ltd.
AChE, acetylcholinesterase; TdT, terminal deoxynucleotidyltransferase; mm, mouse monoclonal antibody; rp, rat polyclonal antibody; MW, microwave pretreatment. a LSAB2 System, Dako Co. b ChemMateDetection kit, Dako Co.
0. No MG symptoms. 1. MG with purely ocular muscle weakness. 2. MG patients with mild generalized weakness, usually with ocular muscle weakness but without bulbar involvement. 3. MG with mild generalized weakness including bulbar involvement with dysarthria, dysphagia and poor mastication. 4. MG patients with moderate generalized weakness, usually with moderate bulbar and respiratory muscle weakness. 5. MG with severe generalized, bulbar and respiratory muscle weakness, or death due to MG-related complications. Besides that grading, the severity of MG was also evaluated by assessment of muscular strength with quantitative evaluation of double vision, ptosis, facial muscles, swallowing, speech after counting, arm and leg outstretching, hand grip, head lifting and vital lung capacity. ‘‘Change in status’’ was evaluated in our cohort 1 year after thymectomy and at the end of follow-up, using the stratification outlined in the Recommendations for MG Clinical Research [9] (for details see Supplementary Table A). ‘‘Postintervention status’’ after thymectomy was recorded according to the criteria defined by MGFA [9] 1 year after thymectomy and at the end of follow-up. Following categories were defined: complete stable remission, pharmacologic remission and minimal manifestations (Supplementary Table A).
3. Results 3.1. Definite polymyositis All 15 patients with definite PM presented with symmetrical proximal muscle weakness affecting predominantly the limb-girdle muscles of lower extremities accompanied by elevated serum levels of CK (ranging from 17.9 to 75.2 lkat/l; normal <6.6 lkat/l). EMG studies revealed myogenic pattern in all the cases accompanied in most of them by small amplitude, polyphasic motor units or fibrillations and positive sharp waves. No clinical, immunological or electrophysiological signs of MG were present. In all PM cases, the endomysial lymphocytic infiltrates were found in muscle biopsy taken from lower limb muscles consisting of CD8+ T lymphocytes (Fig. 1A and C) admixed with few CD68+ macrophages. The necrosis and regeneration of muscle fibers was present scattered throughout in each case. No CD20+ B lymphocytes were noted and the scattered CD4+ T lymphocytes consisted of less than 1% of the inflammatory cells. In 9 cases (60.0%), the CD8+ lymphocytes had invaded the nonnecrotic muscle fibers. All the T lymphocytes in PM cases were CD45RA negative (Fig. 1E). The muscle fibers including normal appearing ones expressed diffusely HLA-I on their surface membranes (Fig. 1G), and moreover, HLA-I was present in cytoplasm of regenerating muscle fibers. Deposits of MAC were identified on the surface of the necrotic fibers. All cases of PM responded well to immunosuppressive therapy with corticosteroids.
2.4. Statistical analysis 3.2. Lymphocytic infiltration in MG Associations between categorical variables were assessed via the Pearson’s chi-square test and associations between categorical and numeric variables were assessed via the Wilcoxon matched-pairs signed-rank test. The analytical work was performed using SPSS (version 10, SPSS Inc.) software. Probability (p) values <0.05 were considered significant.
3.2.1. Clinical features and relationship to the thymus pathology From 179 muscle biopsies obtained during thymectomy in MG patients, lymphocytic infiltration of muscle was seen in 18 (10.1%), all of which were associated with thymomas (no. 1–18; Table 2). Interestingly, this finding was made
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Fig. 1. Comparison of morphological and immunohistological features of lymphocytic infiltrates in polymyositis (PM) and in muscle of a thymomaassociated myasthenia gravis (MG) patient. (A, B) In both the PM and the MG, the lymphocytic infiltrates were localized in the endomysium surrounding the muscle fibers. Note the autoaggressive invasion of lymphocytes into the non-necrotic muscle fibers (asterisk) observed in PM (cryosections, hematoxylin–eosin stain). (C, D) In both PM and MG cases, the lymphocytic infiltrates consisted mostly of CD8+ T lymphocytes (cryosections, immunoperoxidase method, counterstained lightly with hematoxylin). (E, F) The CD8+ lymphocytes in MG were CD45RA positive representing mature but naive T lymphocytes. The lymphocytes in polymyositis were CD45RA negative (cryosections, immunoperoxidase method, counterstained lightly with hematoxylin). (G, H) The overexpression of HLA-I on the surface of muscle fibers was limited to the site of lymphocytic infiltration in MG, the neighboring muscle fibers were completely negative (upper right part). In PM, the overexpression of HLA-I was present even in regions free of inflammatory infiltrate (cryosections, immunoperoxidase method, counterstained lightly with hematoxylin). Bar scale = 50 lm (A, C, E) and 75 lm (B, D, F–H).
only in patients with lymphocyte-rich thymomas, i.e. thymomas of WHO type B (B1–B3), and in one case with a mixed type AB thymoma (B component of which represented a type B2 thymoma). No lymphocytes were found in muscles of MG patients whose thymuses showed follicular hyperplasia (74.3%, n = 133) or simple atrophy (15.6%, n = 28). All the MG patients with lymphocytic infiltrates presented during the follow-up period pure
MG with increased levels of anti-AChR autoantibodies in sera. No additional muscle weakness or myogenic features on EMG were noted. A borderline elevation of serum CK (7.5 lkat/l, normal <6.6 lkat/l) was noted only in one case (no. 13) and the level normalized after the thymectomy. In 7 cases (38.9%) of MG patients with lymphocytic infiltration of muscle and in two thymoma MG patients without lymphocytes in muscle mild blood lymphocytosis
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Table 2 Clinical data and outcome of thymoma patients No. A. Thymoma MG patients with lymphocytes in muscle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1 year Thymoma Sex Age Initial Anti-AChR Ly type (years) MG grade (nmol/l) (· 109/l) CS/PS
Follow-up Last (months) CS/PS
B3 i B2 i B2 i B2 i B2 i B1 i B2 n AB n B1 n B3 i B2 i B1 i B2 i B3 i B2 i B2 i B1 n B2 i
f f m f f m f m m f m m m m m m f f
25 54 44 26 48 58 54 74 76 44 54 51 59 66 68 79 63 70
4 3 3 3 3 4 1 1 1 1 3 1 4 2 2 2 3 3
10.5 18.7 12.8 15.3 7.8 4.6 6.8 7.1 7.8 5.9 2.1 13.5 15.0 5.2 1.5 3.2 9.1 7.1
5.2 n.i. 6.8 n.i. n.i. 6.1 n.i. 4.9 n.i. n.i. n.i. 5.5 n.i. 6.2 n.i. 4.9 n.i. n.i.
U I: MM-2 I: MM-3 I: MM-2 W I: MM-3 I: MM-2 I: MM-2 I: MM-2 I: MM-3 I: MM-1 I: MM-2 I: MM-3 W I: MM-3 I: MM-2 U I: MM-1
32 24 18 24 24 41 41 27 32 52 31 29 77 27 25 13 20 48
I: MM-3 U I: MM-2 U U U U U U I: MM-1 U U U D U U U PM
B. Thymoma MG patients without lymphocytes in muscle 19 B3 i 20 B2 i 21 B1 n 22 AB n 23 An 24 AB n 25 B2 n 26 B2 i 27 B1 n 28 B2 i 29 B2 n 30 B1 n 31 B2 i 32 B2 i 33 An
f f m m f f f f f f f m m f m
32 56 31 47 39 54 60 49 46 33 38 69 58 42 59
3 2 3 4 3 2 2 3 3 3 3 2 2 3 3
1.8 4.0 16.4 10.5 7.3 2.8 13.7 7.7 11.8 6.6 5.8 2.3 4.9 6.6 9.2
n.i. n.i. n.i. n.i. n.i. n.i. n.i. 5.8 n.i. n.i. n.i. n.i. 6.2 n.i. n.i.
I: MM-3 165 I: MM-3 20 W 19 I: MM-3 39 I: MM-2 15 W 14 I: MM-3 54 I: MM-2 13 U 43 I: MM-3 72 I: MM-3 27 I: MM-2 24 I: MM-1 22 I: MM-2 19 I: MM-2 12
PM U U U U U U U U I: MM-2 I: MM-2 U U U U
C. Thymoma patients without muscular involvement 34 35 36 37 38 39
m f f m f f
26 38 52 44 35 51
0 0 0 0 0 0
n.i. n.i. n.i. n.i. n.i. n.i.
n.i. 6.5 n.i. n.i. n.i. n.i.
U U U U U U
U U U U U U
B2 i B3 i An An AB n B1 i
24 37 56 72 19 82
i, invasive; n, non-invasive; m, male; f, female; Anti-AChR, autoantibodies against acetylcholine receptor (normal <0.3 nmol/l); Ly, blood lymphocytosis (normal 1.5–4.6 · 109/l); n.i., not increased; 1 year CS/PS, change of status and postintervention status 1 year after thymectomy; Last CS/PS, change of status and postintervention status at the end of follow-up period (in relation to the 1 year CS/PS); I, improved; U, unchanged; W, worse; D, died; MM1– MM3, minimal manifestations; PM, polymyositis.
(5.2–6.8 · 109/l; normal 1.5–4.6 · 109/l) was noted before surgery (see Table 2) and disappeared thereafter in all of the cases. No increased sedimentation rate or CRP protein level were present. 3.2.2. Lymphocytic infiltrates – morphology and immunophenotype In 15 cases (patients no. 1–15) the morphological pattern of lymphocytic infiltration was indistinguishable from that of PM – endomysial infiltration surrounded the non-
necrotic muscle fibers (Fig. 1B), aside from the fact that the muscle fiber invasion by lymphocytes, which was observed in a proportion of the definite PM cases, was absent in MG. In further 3 cases (no. 16–18) the lymphocytes were scattered in the connective tissue without forming any continuous aggregates and they were recognized by LCA immunohistochemistry. Few isolated necrotic and regenerating muscle fibers were found only in muscle biopsy of patient no. 13, in which mildly elevated CK levels were noted. No muscle fiber necrosis was observed in the
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remaining cases. The infiltrate consisted mostly of CD8+ T lymphocytes (Fig. 1D); CD4+ cells were few (<1% of the inflammatory cells in 10 cases) and B lymphocytes were completely absent. Contrary to PM, the CD68+ macrophages were not present except for scattered cells in a case with muscle fiber necrosis. Membranes of the T lymphocytes in all the thymoma-associated MG muscles were strongly positive in immunohistochemistry with the antiCD45RA (Fig. 1F) demonstrating thereby, that they represent mature but naive (i.e. antigenically not stimulated) T lymphocytes. In patients no. 1–15, the muscle fibers close to the inflammatory infiltrate expressed HLA-I on surface membranes; however, the neighboring muscle fibers were completely negative (Fig. 1H). No pathologic depositions of MAC were identified in the MG muscles. We also examined the relationship of lymphocytes to NMJs. However, the density of NMJs in sternothyroid muscle was very low (<1 NMJ per low-power field) and no spatial relationship of lymphocytic infiltrates to NMJ could be demonstrated (data not shown). 3.3. Lymphocytic infiltration in thymoma patients without MG Two of the six patients with thymoma and no signs of MG or other myopathy (Table 2, nos. 34 and 35) showed lymphocytic infiltration consisting of CD8+CD45RA+ T lymphocytes in sternothyroid muscle and mild blood lymphocytosis in one case (no. 35). All the non-MG thymoma patients remained free of any muscle symptoms during 19– 82 months of follow-up. 3.4. Immunophenotype of lymphocytes in thymomas of MG patients We analyzed the immunophenotype of the lymphocytes of all the resected thymomas. Although the lymphocytes of the type B thymomas in our series consisted predominantly of TdT+CD45RA immature T lymphocytes with cytoplasmic CD3 staining, we observed in all cases that the intratumoral T lymphocytes focally mature into the CD45RA+ phenotype (data not shown). 3.5. Late polymyositis in thymoma MG patients In the thymoma MG patient no. 18 (Table 2), the primary muscle biopsy from sternothyroid muscle during thymectomy revealed scattered CD8+CD45RA+ T lymphocytes in the absence of any myopathic features and the patient’s status improved after thymectomy with minimal MG symptoms. However, almost 4 years later, the patient presented progressive proximal limb-girdle weakness refractory to the pyridostigmine treatment, elevation of CK (up to 30.8 lkat/l) and myogenic EMG pattern with polyphasic potentials. The subsequent biopsy from deltoid muscle disclosed the typical PM pattern, including the presence of CD8+CD45RA T lymphocytes with
autoaggressive muscle fiber invasion and diffuse surface HLA-I positivity of muscle fibers. Similarly, another thymoma MG patient (no. 19, Table 2) presented with the above-mentioned PM signs almost 15 years after reaching the minimal manifestation of MG after thymectomy where there were no lymphocytes observed in the muscle. The subsequent biopsy of vastus femoris muscle revealed typical PM pattern with an infiltration of CD8+CD45RA T lymphocytes and macrophages. The muscular weakness responded well to the intense immunosuppressive treatment in both patients, they recovered from the PM and remained with minimal manifestation of MG symptoms. 3.6. Outcome and statistical analysis The severity of MG at presentation and the outcome after thymectomy (postintervention status and its change) of 18 thymoma MG patients with lymphocytic infiltrate in muscle was compared to the remaining 15 thymoma MG patient (see Table 2). In both groups, neither complete stable remission nor pharmacologic remission was reached and most of the patients presented improved status with minimal manifestation of MG (Supplementary Table B). The minimal MG manifestations retained the myasthenic character; no additional muscle weakness appeared even in cases without further immunosuppression. In two patients from both groups the MG symptoms worsened and one thymoma MG patient from the lymphocyte-infiltration group (no. 14) died 27 months after the thymectomy, but due to reasons unrelated to MG. The statistical analysis revealed that neither the severity nor the clinical course significantly differed among these subgroups – 77% patients of the first group and 80% of the other had improved clinical status, persisting for at least 1 year. Statistical analysis revealed that in the subgroup of thymoma MG patients with lymphocytic infiltration there was an increased frequency of invasive thymomas and increased frequency of WHO type B thymomas (Supplementary Table B). 4. Discussion In MG, in which humoral immune attack upon the NMJ is the main (if not the only) pathogenetic mechanism, there were few hypotheses concerning the enigmatic presence of lymphocytes in affected muscles. The hypothetical role of lymphocytes at the NMJ [4,5] is not supported by our results as well as by another previous report [6]. Concomitant autoimmune inflammation mediated through a disturbance in the cellular immunity, i.e. PM, was proposed as a possible explanation of the lymphocytic infiltrates [7] and more than 20 cases of such an association were published (reviewed by Raschilas et al. [15]). The presence of endomysial infiltration by T lymphocytes in MG muscles (mostly CD8+) argued in support of that theory. However, the clinical features of such cases are often not those of a full-blown PM – some of previously reported
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cases and all in our series did not have additional muscle weakness, relevant elevation of serum CK or myopathic changes by EMG. Therefore, we speculate that PM and lymphocytic infiltration in MG patients with thymomas are separate entities. Our clinical data analysis as well as tissue-based analysis demonstrate that the lymphocytic infiltration in MG differs from that in PM in many aspects, chiefly: the CD8+ T lymphocytes in MG are mature but naive cells (i.e. before activation through presentation of an antigen by antigen-presenting cells) without a propensity for autoaggressive muscle fiber invasion; and, the minimal alteration of the muscle tissue is limited to the site of infiltration with virtual absence of muscle fiber necrosis. This study also reveals the fact that all of the examples of lymphocytic infiltration were associated with lymphocyte-rich thymomas. In our series, the infiltration of MG muscle by naive T lymphocytes was unexpected; moreover, the patients did not present any additional clinical myopathic signs to the underlying MG symptoms. Although the origin of the naive CD8+ T lymphocytes from other lymphoid organs or bone marrow cannot be excluded, there is an increasing evidence that thymomas (especially of WHO type B) are capable of intratumoral thymopoiesis and export of naive mature T cells into the peripheral blood [16,17]. This might be mirrored by the mild lymphocytosis observed in some of our patients, which was also recognized to be a rare phenomenon in MG thymoma patients in the literature [18–22]. Interestingly, all the specific alterations in the blood were reversed by thymectomy in our cases and the above-mentioned reports thus providing a possible explanation for the fact that most of our patients improved in clinical status even if they were not treated for PM by immunosuppression. Still another argument in favor of our hypothesis that lymphocytes in MG muscle may represent only an infiltration by naive T lymphocytes generated by thymomas is provided by our demonstration of collections of mature naive T lymphocytes in non-MG thymoma patients. Although MG appearing later after thymectomy for a thymoma has been recorded in about 1–3% of thymoma patients without muscle involvement at the time of surgery [23], all non-MG thymoma patients in our series were free of muscle symptoms after follow-up of up to 82 months. Although coincidental in nature, the significance of the ‘‘spillover’’ of thymoma-derived lymphocytes and the infiltration of tissues in MG should not be considered minor. The involvement of skeletal muscle leads presumably to subclinical effects; however, the lymphocytic infiltration may even become fatal when affecting the contractile function of heart [24]. Moreover, beside the decreased frequency of intrathymic regulatory T cells that promotes the non-toleragenic intratumoral thymopoeisis in thymoma MG patients [25], it has been suggested that development of MG might be associated with the efficiency of WHO type B thymomas to produce and export naive CD4+ T cells [26].
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In unusual cases of MG, a true autoimmune PM develops adding to the weakness already caused by the neuromuscular transmission defect (as reviewed by Aarli [7]), such as was observed in two of our patients. In one case there was no inflammation in muscle at the time of thymectomy for thymoma followed by an interval of almost 15 years before developing classical PM. The other patient in our series presented naive mature T lymphocytes in muscle at the time of thymectomy that switched into antigenically stimulated CD8+CD45RA lymphocytes 4 years later when presenting clinically as a typical PM. This phenomenon could be explained by the fact that the abnormal thymopoiesis occurring within thymomas alters the peripheral T cell repertoire, thereby generating greater autoantigen-specific potential [16,26]. Following sensitization in the periphery, the initially naive CD8+ T lymphocytes might be the source of a later lymphocytic attack against the skeletal muscle fibers. Taken together, we have demonstrated that lymphocytes infiltrate muscles of MG patients more frequently than expected, perhaps due to the fact that patients with pure MG are not examined by muscle biopsy routinely. It represents rather a consequence of the ‘‘spillover’’ and infiltration of thymoma-derived mature naive T cells than a true cell-mediated autoimmune inflammation, since the infiltration differs in both clinical features and immunophenotype from classical PM. In the vast majority of typical cases, PM can be distinguished from MG based on clinical and electrophysiological findings. In questionable cases, however, the patient might be referred to muscle biopsy, where the finding of endomysial CD8+ lymphocytes in muscle biopsies (especially in association with blood lymphocytosis) should not be overdiagnosed and treated as PM, but should (after demonstrating the CD45RA positivity of the lymphocytes) suggest the diagnosis of thymomaassociated disorder and appropriate imaging studies should be performed to exclude a thymic mass. Acknowledgement The authors thank Adrian Cibula and Iva Simkova for excellent technical assistance in histological procedures. This study was supported by the Grant of Ministry of Health of the Czech Republic No. IGA MZCR NR/ 8924-3 and by the Research Project VZ FNM 00064203. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.nmd.2007. 05.010. References [1] Romi F, Gilhus NE, Aarli JA. Myasthenia gravis: clinical, immunological, and therapeutic advances. Acta Neurol Scand 2005;111:134–41.
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[15] Raschilas F, Mouthon L, Andre MH, et al. Concomitant polymyositis and myasthenia gravis reveal malignant thymoma. A case report and review of the literature. Ann Med Interne (Paris) 1999;150:370–3. [16] Hoffacker V, Schultz A, Tiesinga JJ, et al. Thymomas alter the T-cell subset composition in the blood: a potential mechanism for thymoma-associated autoimmune disease. Blood 2000;96:3872–9. [17] Marx A, Muller-Hermelink HK, Strobel P. The role of thymomas in the development of myasthenia gravis. Ann NY Acad Sci 2003;998:223–36. [18] Barton AD. T-cell lymphocytosis associated with lymphocyte-rich thymoma. Cancer 1997;80:1409–17. [19] Cranney A, Markman S, Lach B, Karsh J. Polymyositis in a patient with thymoma and T cell lymphocytosis. J Rheumatol 1997;24:1413–6. [20] Doll DC, Landreneau RJ, List AF. Malignant thymoma associated with peripheral T-cell lymphocytosis. Med Pediatr Oncol 1991;19:496–8. [21] Medeiros LJ, Bhagat SK, Naylor P, et al. Malignant thymoma associated with T-cell lymphocytosis. A case report with immunophenotypic and gene rearrangement analysis. Arch Pathol Lab Med 1993;117:279–83. [22] Otton SH, Standen GR, Ormerod IE. T cell lymphocytosis associated with polymyositis, myasthenia gravis and thymoma. Clin Lab Haematol 2000;22:307–8. [23] Kondo K, Monden Y. Myasthenia gravis appearing after thymectomy for thymoma. Eur J Cardiothorac Surg 2005;28:22–5. [24] Reznik M. Two cases of myasthenic syndrome with thymoma, polymyositis, myocarditis, and thyroiditis. J Neurol Sci 1974;22:341–51. [25] Luther C, Poeschel S, Varga M, Melms A, Tolosa E. Decreased frequency of intrathymic regulatory T cells in patients with myasthenia-associated thymoma. J Neuroimmunol 2005;164:124–8. [26] Strobel P, Helmreich M, Menioudakis G, et al. Paraneoplastic myasthenia gravis correlates with generation of mature naive CD4(+) T cells in thymomas. Blood 2002;100:159–66.
Muscle lymphocytic infiltrates in thymoma-associated myasthenia gravis are phenotypically different from those in polymyositis Josef Zamecnik a
d
a,*
, Dan Vesely a, Branislav Jakubicka a, Libuse Simkova b, Jiri Pitha c, Jan Schutzner d, Radim Mazanec e, Hannes Vogel f
Department of Pathology and Molecular Medicine, Charles University, 2nd Medical Faculty and Faculty Hospital Motol, V Uvalu 84, 150 06 Prague, Czech Republic b Department of Neurology, Charles University, 1st Medical Faculty, Prague, Czech Republic c Department of Neurology, Charles University, 3rd Medical Faculty, Prague, Czech Republic 3rd Department of Surgery, Charles University, 1st Medical Faculty and Faculty Hospital Motol, Prague, Czech Republic e Department of Neurology, Charles University, 2nd Medical Faculty, Prague, Czech Republic f Department of Pathology, Stanford University School of Medicine, Palo Alto, CA, USA Received 10 April 2007; received in revised form 21 May 2007; accepted 25 May 2007
Abstract The aim of the study is to provide evidence that the lymphocytic infiltration of myasthenia gravis (MG) muscle do not represent a true autoimmune myositis, rather an infiltration by naive lymphocytes derived from lymphocyte-rich thymomas. Muscle biopsies from 179 patients with pure MG, 6 thymoma patients without MG and 15 patients with definite polymyositis were analyzed. In 18 patients with MG (all associated with lymphocyte-rich thymomas) and in two thymoma patients without MG, lymphocytic infiltrates were identified in muscles. By use of immunohistochemistry, we demonstrated that the lymphocytes in MG differ from those in polymyositis, being mature but in contrast to polymyositis naive CD45RA+ T lymphocytes. We suggest that the lymphocytic infiltrates in patients with MG and thymoma represent an infiltration of muscle by thymoma-derived mature but naive T cells. The finding of CD8+CD45RA+ lymphocytes in muscle may signify an underlying thymoma and should not be misdiagnosed as polymyositis. 2007 Elsevier B.V. All rights reserved. Keywords: Myasthenia gravis; Polymyositis; Thymoma
1. Introduction The principal disease mechanism in myasthenia gravis (MG) is a humoral immune attack upon the acetylcholine receptor (AChR) leading to the characteristic defect of neuromuscular transmission [1,2]. Focal collections of mononuclear cells in MG muscles, so-called ‘‘lymphorrhages’’, were reported over 100 years ago [3]; however, their significance and pathogenic role has been a challenging issue in
*
Corresponding author. Tel.: +420 224 435 635; fax: +420 224 435 620. E-mail address: [email protected] (J. Zamecnik).
0960-8966/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2007.05.010
neuropathology for decades. The proposed role in causing MG by acting at the neuromuscular junction (NMJ) [4,5] could not be subsequently confirmed [6] and the presence of lymphocytes has been often interpreted as a coincidental inflammatory myopathy – polymyositis (PM). On the other hand, an increasing body of evidence suggests that the ‘‘PM’’ occurring in MG patients is clinically not of the same type as a classical PM [7], aside from the fact that PM is traditionally regarded as a disturbance of cellular immunity in striated muscle [8]. Our interest in this problem began with the case of a patient with the ambiguous presentation of muscle weakness with non-conclusive initial EMG findings, borderline
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serum levels of anti-AChR autoantibodies and normal serum levels of creatine kinase (CK), referred for a muscle biopsy. Although the infiltration of muscle by CD8+ lymphocytes was interpreted as a PM, the patient soon developed classic MG; a subsequent CT scan of mediastinum revealed a thymic tumor and after thymectomy the patient remained treated with pyridostigmin without immunosuppression and his status improved. This case prompted our systematic study of lymphocytic infiltrates in muscle tissue from MG patients. We present results indicating that true autoimmune PM may rarely develop in MG patients, but that it should be distinguished from the more frequent occurrence, i.e. the infiltration by naive lymphocytes derived from lymphocyte-rich thymomas. 2. Materials and methods 2.1. Patients 179 thymectomized MG patients (78 males and 101 females, median age 49 years) with available follow-up data for at least 1 year after thymectomy were retrospectively included in the study. The diagnosis of MG was based on a typical clinical pattern, a positive edrophonium test and neurophysiological investigations with decrement at repetitive stimulation and/or increased jitter at single-fiber EMG [2], in addition to the presence of an increased serum level of anti-AChR antibodies in the vast majority of cases (91.1%, n = 163). All patients underwent CT scan of the anterior mediastinum, which showed a suspicious thymic tumor in 33 cases (18.4%). In each case, the presence of the thymoma was verified by subsequent surgery and the diagnosis was confirmed by the histopathological examination. The clinical data were collected both at the time of initial diagnosis, shortly preoperatively, then one year after thymectomy and onwards each year to the end of followup (from 12 up to 165 months; median ± standard deviation, 27 ± 4.9 months). We recorded clinical symptoms, serum levels of CK and transaminases, erythrocyte sedimentation rate, C-reactive protein (CRP) level and blood cell counts. A standard transsternal thymectomy was performed in all the patients (T-3a according to the Myasthenia Gravis Foundation of America (MGFA) thymectomy classification [9]). All thymuses were examined histologically. Thymomas were typed according to the recent WHO classification of thymomas [10] into following categories: type A (spindle cell or medullary thymoma), type B (lymphocytes-rich, cortical thymoma) and mixed type AB thymoma. No other thymic tumors were observed in our series. The immunophenotype of the lymphocytes in thymomas was analyzed using the same antibodies as in the muscle (see below). Except for MG patients, muscle biopsies of 6 thymoma patients (median age 41 years) without any signs of muscle involvement at the time of surgery with available follow-up data entered into the study. Muscle biopsies of 15 patients (median age 57 years) with definite isolated PM based on the clinical findings,
EMG and confirmed by muscle biopsy with significant response to immunosuppressive therapy were examined as a control group. 2.2. Muscle biopsy – histology, histochemistry and immunohistology An excision from the sternothyroid muscle (approx. 10 · 5 · 5 mm in size) was obtained from all the thymectomized patients from the margin of the operational field at the onset of thymectomy (informed consent of the patient was obtained in each case). In patients with PM lower limb muscles were biopsied. Muscle tissues were snap frozen in isopentane (2-methylbutane, Sigma–Aldrich Co.) cooled in liquid nitrogen. Cryosections were examined by routine hematoxylin–eosin (H&E) staining and a conventional spectrum of histochemical reactions (described elsewhere [11]), from which the visualization of neuromuscular junctions using reactions for non-specific esterase was relevant for the study. In each case, we searched for lymphocytes in the serial cryosections by use of both the routine H&E stain and the immunohistochemical reactions with anti-CD45 (leukocyte common antigen, LCA) antibody. Provided that lymphocytes were found, the contiguous sections were subjected to immunohistochemistry for establishing their phenotype using antibodies against the antigens CD20, CD3, CD8, CD4 and TdT (terminal deoxynucleotidyltransferase). The CD45RA antigen (present on B lymphocytes and on mature but naive T lymphocytes) was also investigated. The number of macrophages was assessed using the anti-CD68 immunohistochemistry. Helpful tools in diagnosis of inflammatory myopathies, i.e. expression of HLA-I antigens and of membrane attack complex of complement (MAC, C5b-9) [12], were also examined. In addition to the non-specific esterase, immunohistochemistry for acetylcholinesterase was performed for localization of NMJs. Further details concerning the antibodies used in the study are indicated in Table 1. Following blocking of endogenous peroxidase activity, 4 lm thick cryosections were incubated overnight at 4C with appropriate primary antibodies. The antigen–antibody complexes were visualized by biotin–streptavidin detection systems (LSAB2 System or ChemMate Detection kit, Dako Co.). Chromogenic development was performed using 3,3 0 -diaminobenzidine (DAB, Fluka Chemie GmbH.). All sections were counterstained slightly with Harris’ hematoxylin. Positive and negative controls were provided with each assay. 2.3. Evaluation of MG severity and the outcome of MG patients Since the recently recommended Quantitative MG score for Disease Severity (QMGSDS) [9] could not be obtained from all patients at the onset of the disease in this retrospective study, the initial severity of MG was graded using a modified Osserman’s scale [13,14]:
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Table 1 Antibodies, dilutions, pretreatment and detection kits used for the immunohistochemical studies Antigen
Antibody; clone
Source
Dilution, pretreatment, detection kit
CD45 (LCA) CD20 CD3 CD4 CD8 CD45RA CD68 AChE HLA-I C5b-9 (MAC) TdT
mm; 2B11 + PD7/26 mm; L26 rp; NCL-CD3p mm; 4B12 mm; C8/144B mm; 4KB5 mm; PG-M1 mm; HR2 mm; W6/32 mm; aE11 mm, SEN28
Dako, Co. Dako, Co. Novocastra, Novocastra, Dako, Co. Dako, Co. Dako, Co. Novocastra, Dako, Co. Dako, Co. Novocastra,
1:1000, MWa 1:300, MWa 1:200, MWb 1:20, MWb 1:200, MWb 1:75, MWa 1:100, MWa 1:40a 1:900a 1:100a 1:100, MWa
Ltd. Ltd.
Ltd.
Ltd.
AChE, acetylcholinesterase; TdT, terminal deoxynucleotidyltransferase; mm, mouse monoclonal antibody; rp, rat polyclonal antibody; MW, microwave pretreatment. a LSAB2 System, Dako Co. b ChemMateDetection kit, Dako Co.
0. No MG symptoms. 1. MG with purely ocular muscle weakness. 2. MG patients with mild generalized weakness, usually with ocular muscle weakness but without bulbar involvement. 3. MG with mild generalized weakness including bulbar involvement with dysarthria, dysphagia and poor mastication. 4. MG patients with moderate generalized weakness, usually with moderate bulbar and respiratory muscle weakness. 5. MG with severe generalized, bulbar and respiratory muscle weakness, or death due to MG-related complications. Besides that grading, the severity of MG was also evaluated by assessment of muscular strength with quantitative evaluation of double vision, ptosis, facial muscles, swallowing, speech after counting, arm and leg outstretching, hand grip, head lifting and vital lung capacity. ‘‘Change in status’’ was evaluated in our cohort 1 year after thymectomy and at the end of follow-up, using the stratification outlined in the Recommendations for MG Clinical Research [9] (for details see Supplementary Table A). ‘‘Postintervention status’’ after thymectomy was recorded according to the criteria defined by MGFA [9] 1 year after thymectomy and at the end of follow-up. Following categories were defined: complete stable remission, pharmacologic remission and minimal manifestations (Supplementary Table A).
3. Results 3.1. Definite polymyositis All 15 patients with definite PM presented with symmetrical proximal muscle weakness affecting predominantly the limb-girdle muscles of lower extremities accompanied by elevated serum levels of CK (ranging from 17.9 to 75.2 lkat/l; normal <6.6 lkat/l). EMG studies revealed myogenic pattern in all the cases accompanied in most of them by small amplitude, polyphasic motor units or fibrillations and positive sharp waves. No clinical, immunological or electrophysiological signs of MG were present. In all PM cases, the endomysial lymphocytic infiltrates were found in muscle biopsy taken from lower limb muscles consisting of CD8+ T lymphocytes (Fig. 1A and C) admixed with few CD68+ macrophages. The necrosis and regeneration of muscle fibers was present scattered throughout in each case. No CD20+ B lymphocytes were noted and the scattered CD4+ T lymphocytes consisted of less than 1% of the inflammatory cells. In 9 cases (60.0%), the CD8+ lymphocytes had invaded the nonnecrotic muscle fibers. All the T lymphocytes in PM cases were CD45RA negative (Fig. 1E). The muscle fibers including normal appearing ones expressed diffusely HLA-I on their surface membranes (Fig. 1G), and moreover, HLA-I was present in cytoplasm of regenerating muscle fibers. Deposits of MAC were identified on the surface of the necrotic fibers. All cases of PM responded well to immunosuppressive therapy with corticosteroids.
2.4. Statistical analysis 3.2. Lymphocytic infiltration in MG Associations between categorical variables were assessed via the Pearson’s chi-square test and associations between categorical and numeric variables were assessed via the Wilcoxon matched-pairs signed-rank test. The analytical work was performed using SPSS (version 10, SPSS Inc.) software. Probability (p) values <0.05 were considered significant.
3.2.1. Clinical features and relationship to the thymus pathology From 179 muscle biopsies obtained during thymectomy in MG patients, lymphocytic infiltration of muscle was seen in 18 (10.1%), all of which were associated with thymomas (no. 1–18; Table 2). Interestingly, this finding was made
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Fig. 1. Comparison of morphological and immunohistological features of lymphocytic infiltrates in polymyositis (PM) and in muscle of a thymomaassociated myasthenia gravis (MG) patient. (A, B) In both the PM and the MG, the lymphocytic infiltrates were localized in the endomysium surrounding the muscle fibers. Note the autoaggressive invasion of lymphocytes into the non-necrotic muscle fibers (asterisk) observed in PM (cryosections, hematoxylin–eosin stain). (C, D) In both PM and MG cases, the lymphocytic infiltrates consisted mostly of CD8+ T lymphocytes (cryosections, immunoperoxidase method, counterstained lightly with hematoxylin). (E, F) The CD8+ lymphocytes in MG were CD45RA positive representing mature but naive T lymphocytes. The lymphocytes in polymyositis were CD45RA negative (cryosections, immunoperoxidase method, counterstained lightly with hematoxylin). (G, H) The overexpression of HLA-I on the surface of muscle fibers was limited to the site of lymphocytic infiltration in MG, the neighboring muscle fibers were completely negative (upper right part). In PM, the overexpression of HLA-I was present even in regions free of inflammatory infiltrate (cryosections, immunoperoxidase method, counterstained lightly with hematoxylin). Bar scale = 50 lm (A, C, E) and 75 lm (B, D, F–H).
only in patients with lymphocyte-rich thymomas, i.e. thymomas of WHO type B (B1–B3), and in one case with a mixed type AB thymoma (B component of which represented a type B2 thymoma). No lymphocytes were found in muscles of MG patients whose thymuses showed follicular hyperplasia (74.3%, n = 133) or simple atrophy (15.6%, n = 28). All the MG patients with lymphocytic infiltrates presented during the follow-up period pure
MG with increased levels of anti-AChR autoantibodies in sera. No additional muscle weakness or myogenic features on EMG were noted. A borderline elevation of serum CK (7.5 lkat/l, normal <6.6 lkat/l) was noted only in one case (no. 13) and the level normalized after the thymectomy. In 7 cases (38.9%) of MG patients with lymphocytic infiltration of muscle and in two thymoma MG patients without lymphocytes in muscle mild blood lymphocytosis
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Table 2 Clinical data and outcome of thymoma patients No. A. Thymoma MG patients with lymphocytes in muscle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1 year Thymoma Sex Age Initial Anti-AChR Ly type (years) MG grade (nmol/l) (· 109/l) CS/PS
Follow-up Last (months) CS/PS
B3 i B2 i B2 i B2 i B2 i B1 i B2 n AB n B1 n B3 i B2 i B1 i B2 i B3 i B2 i B2 i B1 n B2 i
f f m f f m f m m f m m m m m m f f
25 54 44 26 48 58 54 74 76 44 54 51 59 66 68 79 63 70
4 3 3 3 3 4 1 1 1 1 3 1 4 2 2 2 3 3
10.5 18.7 12.8 15.3 7.8 4.6 6.8 7.1 7.8 5.9 2.1 13.5 15.0 5.2 1.5 3.2 9.1 7.1
5.2 n.i. 6.8 n.i. n.i. 6.1 n.i. 4.9 n.i. n.i. n.i. 5.5 n.i. 6.2 n.i. 4.9 n.i. n.i.
U I: MM-2 I: MM-3 I: MM-2 W I: MM-3 I: MM-2 I: MM-2 I: MM-2 I: MM-3 I: MM-1 I: MM-2 I: MM-3 W I: MM-3 I: MM-2 U I: MM-1
32 24 18 24 24 41 41 27 32 52 31 29 77 27 25 13 20 48
I: MM-3 U I: MM-2 U U U U U U I: MM-1 U U U D U U U PM
B. Thymoma MG patients without lymphocytes in muscle 19 B3 i 20 B2 i 21 B1 n 22 AB n 23 An 24 AB n 25 B2 n 26 B2 i 27 B1 n 28 B2 i 29 B2 n 30 B1 n 31 B2 i 32 B2 i 33 An
f f m m f f f f f f f m m f m
32 56 31 47 39 54 60 49 46 33 38 69 58 42 59
3 2 3 4 3 2 2 3 3 3 3 2 2 3 3
1.8 4.0 16.4 10.5 7.3 2.8 13.7 7.7 11.8 6.6 5.8 2.3 4.9 6.6 9.2
n.i. n.i. n.i. n.i. n.i. n.i. n.i. 5.8 n.i. n.i. n.i. n.i. 6.2 n.i. n.i.
I: MM-3 165 I: MM-3 20 W 19 I: MM-3 39 I: MM-2 15 W 14 I: MM-3 54 I: MM-2 13 U 43 I: MM-3 72 I: MM-3 27 I: MM-2 24 I: MM-1 22 I: MM-2 19 I: MM-2 12
PM U U U U U U U U I: MM-2 I: MM-2 U U U U
C. Thymoma patients without muscular involvement 34 35 36 37 38 39
m f f m f f
26 38 52 44 35 51
0 0 0 0 0 0
n.i. n.i. n.i. n.i. n.i. n.i.
n.i. 6.5 n.i. n.i. n.i. n.i.
U U U U U U
U U U U U U
B2 i B3 i An An AB n B1 i
24 37 56 72 19 82
i, invasive; n, non-invasive; m, male; f, female; Anti-AChR, autoantibodies against acetylcholine receptor (normal <0.3 nmol/l); Ly, blood lymphocytosis (normal 1.5–4.6 · 109/l); n.i., not increased; 1 year CS/PS, change of status and postintervention status 1 year after thymectomy; Last CS/PS, change of status and postintervention status at the end of follow-up period (in relation to the 1 year CS/PS); I, improved; U, unchanged; W, worse; D, died; MM1– MM3, minimal manifestations; PM, polymyositis.
(5.2–6.8 · 109/l; normal 1.5–4.6 · 109/l) was noted before surgery (see Table 2) and disappeared thereafter in all of the cases. No increased sedimentation rate or CRP protein level were present. 3.2.2. Lymphocytic infiltrates – morphology and immunophenotype In 15 cases (patients no. 1–15) the morphological pattern of lymphocytic infiltration was indistinguishable from that of PM – endomysial infiltration surrounded the non-
necrotic muscle fibers (Fig. 1B), aside from the fact that the muscle fiber invasion by lymphocytes, which was observed in a proportion of the definite PM cases, was absent in MG. In further 3 cases (no. 16–18) the lymphocytes were scattered in the connective tissue without forming any continuous aggregates and they were recognized by LCA immunohistochemistry. Few isolated necrotic and regenerating muscle fibers were found only in muscle biopsy of patient no. 13, in which mildly elevated CK levels were noted. No muscle fiber necrosis was observed in the
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remaining cases. The infiltrate consisted mostly of CD8+ T lymphocytes (Fig. 1D); CD4+ cells were few (<1% of the inflammatory cells in 10 cases) and B lymphocytes were completely absent. Contrary to PM, the CD68+ macrophages were not present except for scattered cells in a case with muscle fiber necrosis. Membranes of the T lymphocytes in all the thymoma-associated MG muscles were strongly positive in immunohistochemistry with the antiCD45RA (Fig. 1F) demonstrating thereby, that they represent mature but naive (i.e. antigenically not stimulated) T lymphocytes. In patients no. 1–15, the muscle fibers close to the inflammatory infiltrate expressed HLA-I on surface membranes; however, the neighboring muscle fibers were completely negative (Fig. 1H). No pathologic depositions of MAC were identified in the MG muscles. We also examined the relationship of lymphocytes to NMJs. However, the density of NMJs in sternothyroid muscle was very low (<1 NMJ per low-power field) and no spatial relationship of lymphocytic infiltrates to NMJ could be demonstrated (data not shown). 3.3. Lymphocytic infiltration in thymoma patients without MG Two of the six patients with thymoma and no signs of MG or other myopathy (Table 2, nos. 34 and 35) showed lymphocytic infiltration consisting of CD8+CD45RA+ T lymphocytes in sternothyroid muscle and mild blood lymphocytosis in one case (no. 35). All the non-MG thymoma patients remained free of any muscle symptoms during 19– 82 months of follow-up. 3.4. Immunophenotype of lymphocytes in thymomas of MG patients We analyzed the immunophenotype of the lymphocytes of all the resected thymomas. Although the lymphocytes of the type B thymomas in our series consisted predominantly of TdT+CD45RA immature T lymphocytes with cytoplasmic CD3 staining, we observed in all cases that the intratumoral T lymphocytes focally mature into the CD45RA+ phenotype (data not shown). 3.5. Late polymyositis in thymoma MG patients In the thymoma MG patient no. 18 (Table 2), the primary muscle biopsy from sternothyroid muscle during thymectomy revealed scattered CD8+CD45RA+ T lymphocytes in the absence of any myopathic features and the patient’s status improved after thymectomy with minimal MG symptoms. However, almost 4 years later, the patient presented progressive proximal limb-girdle weakness refractory to the pyridostigmine treatment, elevation of CK (up to 30.8 lkat/l) and myogenic EMG pattern with polyphasic potentials. The subsequent biopsy from deltoid muscle disclosed the typical PM pattern, including the presence of CD8+CD45RA T lymphocytes with
autoaggressive muscle fiber invasion and diffuse surface HLA-I positivity of muscle fibers. Similarly, another thymoma MG patient (no. 19, Table 2) presented with the above-mentioned PM signs almost 15 years after reaching the minimal manifestation of MG after thymectomy where there were no lymphocytes observed in the muscle. The subsequent biopsy of vastus femoris muscle revealed typical PM pattern with an infiltration of CD8+CD45RA T lymphocytes and macrophages. The muscular weakness responded well to the intense immunosuppressive treatment in both patients, they recovered from the PM and remained with minimal manifestation of MG symptoms. 3.6. Outcome and statistical analysis The severity of MG at presentation and the outcome after thymectomy (postintervention status and its change) of 18 thymoma MG patients with lymphocytic infiltrate in muscle was compared to the remaining 15 thymoma MG patient (see Table 2). In both groups, neither complete stable remission nor pharmacologic remission was reached and most of the patients presented improved status with minimal manifestation of MG (Supplementary Table B). The minimal MG manifestations retained the myasthenic character; no additional muscle weakness appeared even in cases without further immunosuppression. In two patients from both groups the MG symptoms worsened and one thymoma MG patient from the lymphocyte-infiltration group (no. 14) died 27 months after the thymectomy, but due to reasons unrelated to MG. The statistical analysis revealed that neither the severity nor the clinical course significantly differed among these subgroups – 77% patients of the first group and 80% of the other had improved clinical status, persisting for at least 1 year. Statistical analysis revealed that in the subgroup of thymoma MG patients with lymphocytic infiltration there was an increased frequency of invasive thymomas and increased frequency of WHO type B thymomas (Supplementary Table B). 4. Discussion In MG, in which humoral immune attack upon the NMJ is the main (if not the only) pathogenetic mechanism, there were few hypotheses concerning the enigmatic presence of lymphocytes in affected muscles. The hypothetical role of lymphocytes at the NMJ [4,5] is not supported by our results as well as by another previous report [6]. Concomitant autoimmune inflammation mediated through a disturbance in the cellular immunity, i.e. PM, was proposed as a possible explanation of the lymphocytic infiltrates [7] and more than 20 cases of such an association were published (reviewed by Raschilas et al. [15]). The presence of endomysial infiltration by T lymphocytes in MG muscles (mostly CD8+) argued in support of that theory. However, the clinical features of such cases are often not those of a full-blown PM – some of previously reported
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cases and all in our series did not have additional muscle weakness, relevant elevation of serum CK or myopathic changes by EMG. Therefore, we speculate that PM and lymphocytic infiltration in MG patients with thymomas are separate entities. Our clinical data analysis as well as tissue-based analysis demonstrate that the lymphocytic infiltration in MG differs from that in PM in many aspects, chiefly: the CD8+ T lymphocytes in MG are mature but naive cells (i.e. before activation through presentation of an antigen by antigen-presenting cells) without a propensity for autoaggressive muscle fiber invasion; and, the minimal alteration of the muscle tissue is limited to the site of infiltration with virtual absence of muscle fiber necrosis. This study also reveals the fact that all of the examples of lymphocytic infiltration were associated with lymphocyte-rich thymomas. In our series, the infiltration of MG muscle by naive T lymphocytes was unexpected; moreover, the patients did not present any additional clinical myopathic signs to the underlying MG symptoms. Although the origin of the naive CD8+ T lymphocytes from other lymphoid organs or bone marrow cannot be excluded, there is an increasing evidence that thymomas (especially of WHO type B) are capable of intratumoral thymopoiesis and export of naive mature T cells into the peripheral blood [16,17]. This might be mirrored by the mild lymphocytosis observed in some of our patients, which was also recognized to be a rare phenomenon in MG thymoma patients in the literature [18–22]. Interestingly, all the specific alterations in the blood were reversed by thymectomy in our cases and the above-mentioned reports thus providing a possible explanation for the fact that most of our patients improved in clinical status even if they were not treated for PM by immunosuppression. Still another argument in favor of our hypothesis that lymphocytes in MG muscle may represent only an infiltration by naive T lymphocytes generated by thymomas is provided by our demonstration of collections of mature naive T lymphocytes in non-MG thymoma patients. Although MG appearing later after thymectomy for a thymoma has been recorded in about 1–3% of thymoma patients without muscle involvement at the time of surgery [23], all non-MG thymoma patients in our series were free of muscle symptoms after follow-up of up to 82 months. Although coincidental in nature, the significance of the ‘‘spillover’’ of thymoma-derived lymphocytes and the infiltration of tissues in MG should not be considered minor. The involvement of skeletal muscle leads presumably to subclinical effects; however, the lymphocytic infiltration may even become fatal when affecting the contractile function of heart [24]. Moreover, beside the decreased frequency of intrathymic regulatory T cells that promotes the non-toleragenic intratumoral thymopoeisis in thymoma MG patients [25], it has been suggested that development of MG might be associated with the efficiency of WHO type B thymomas to produce and export naive CD4+ T cells [26].
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In unusual cases of MG, a true autoimmune PM develops adding to the weakness already caused by the neuromuscular transmission defect (as reviewed by Aarli [7]), such as was observed in two of our patients. In one case there was no inflammation in muscle at the time of thymectomy for thymoma followed by an interval of almost 15 years before developing classical PM. The other patient in our series presented naive mature T lymphocytes in muscle at the time of thymectomy that switched into antigenically stimulated CD8+CD45RA lymphocytes 4 years later when presenting clinically as a typical PM. This phenomenon could be explained by the fact that the abnormal thymopoiesis occurring within thymomas alters the peripheral T cell repertoire, thereby generating greater autoantigen-specific potential [16,26]. Following sensitization in the periphery, the initially naive CD8+ T lymphocytes might be the source of a later lymphocytic attack against the skeletal muscle fibers. Taken together, we have demonstrated that lymphocytes infiltrate muscles of MG patients more frequently than expected, perhaps due to the fact that patients with pure MG are not examined by muscle biopsy routinely. It represents rather a consequence of the ‘‘spillover’’ and infiltration of thymoma-derived mature naive T cells than a true cell-mediated autoimmune inflammation, since the infiltration differs in both clinical features and immunophenotype from classical PM. In the vast majority of typical cases, PM can be distinguished from MG based on clinical and electrophysiological findings. In questionable cases, however, the patient might be referred to muscle biopsy, where the finding of endomysial CD8+ lymphocytes in muscle biopsies (especially in association with blood lymphocytosis) should not be overdiagnosed and treated as PM, but should (after demonstrating the CD45RA positivity of the lymphocytes) suggest the diagnosis of thymomaassociated disorder and appropriate imaging studies should be performed to exclude a thymic mass. Acknowledgement The authors thank Adrian Cibula and Iva Simkova for excellent technical assistance in histological procedures. This study was supported by the Grant of Ministry of Health of the Czech Republic No. IGA MZCR NR/ 8924-3 and by the Research Project VZ FNM 00064203. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.nmd.2007. 05.010. References [1] Romi F, Gilhus NE, Aarli JA. Myasthenia gravis: clinical, immunological, and therapeutic advances. Acta Neurol Scand 2005;111:134–41.
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